Method for rapid purification of umbilical cord MSC exosomes and application thereof in preparation of drugs for treating chronic obstructive pulmonary disease

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a rapid purification method for umbilical cord MSC exosomes and their application in the preparation of drugs for treating COPD. This invention discloses a method for rapidly purifying umbilical cord mesenchymal stromal cell (MSC) exosomes and a pharmaceutical composition comprising umbilical cord MSC exosomes and yamosaponin. In this pharmaceutical composition, the umbilical cord MSC exosomes and yamosaponin synergistically enhance the dynamic compliance of COPD rats, reduce airway resistance, decrease pulmonary inflammation, and repair pulmonary dysfunction. This invention provides a new technical solution for the preparation of drugs for treating or improving COPD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a rapid purification method for umbilical cord MSC exosomes and their application in the preparation of drugs for treating COPD. Background Technology

[0002] Exosomes are lipid bilayer vesicles with a diameter of 30-150 nm secreted by cells. They carry bioactive substances such as proteins, mRNA, microRNA, and lipids, playing a central role in the body's immune response, cell communication, tissue homeostasis, and regeneration. Umbilical cord-derived exosomes, such as umbilical cord mesenchymal matrix cell exosomes, transport bioactive substances to target cells through membrane fusion or endocytosis, enabling intercellular communication and regulating target cell function. They possess multiple functions, including inhibiting inflammatory responses, immune regulation, angiogenesis, and tissue repair. Compared to stem cells, exosomes exhibit superior stability, safety, rapid onset of action, and ease of preservation, making them an emerging strategy in the field of cell-free therapy.

[0003] Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease with a progressively increasing mortality rate in my country. Its core pathological mechanism is closely related to the chronic inflammatory response of the airways and lung tissue to harmful particles or gases. The disease is characterized by incompletely reversible airflow limitation, which progresses over time. Pathological features include chronic inflammatory infiltration of the airways, lung parenchyma, and pulmonary vessels, with a significant increase in alveolar macrophages, T lymphocytes, and neutrophils in different lung sites. Activated inflammatory cells continuously release inflammatory mediators such as IL-6 and TNF-α, leading to repeated abnormal damage-repair cycles in the airway walls. This ultimately results in small airway remodeling, alveolar wall rupture, and decreased lung elastic recoil, leading to persistent airflow limitation and becoming a major cause of death in chronic respiratory diseases.

[0004] Although exosomes possess a variety of biological activities, there are no reports of the application of umbilical cord MSC exosome-related compositions in chronic obstructive pulmonary disease (COPD). In addition, existing exosome extraction methods suffer from problems such as damaging the exosome membrane structure, leading to cytokine leakage and triggering a cytokine storm in vivo, as well as low extraction and purification efficiency, which limit the clinical application of exosomes. Therefore, researching exosome purification methods to obtain high-purity exosomes and then developing exosome compositions with therapeutic effects on COPD is of great research value. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention optimizes existing purification methods for umbilical cord MSC exosomes, establishes a rapid purification method for umbilical cord MSC exosomes, screens for naturally derived active molecules that can be synergistically combined with umbilical cord MSC exosomes to prepare umbilical cord MSC exosome compositions, evaluates the role of these compositions in the treatment of COPD, and obtains an umbilical cord MSC exosome composition with therapeutic effects on COPD, thus providing more technical solutions for the treatment of COPD.

[0006] On one hand, the present invention provides a pharmaceutical composition for treating chronic obstructive pulmonary disease, the pharmaceutical composition comprising umbilical cord MSC exosomes and yam saponins.

[0007] Further, the pharmaceutical composition, by weight, comprises 9-30 parts of umbilical cord MSC exosomes and 0.001-0.011 parts of yam saponin.

[0008] Further, the pharmaceutical composition, by weight, comprises 27-17 parts of umbilical cord MSC exosomes and 0.002-0.005 parts of yam saponin.

[0009] Furthermore, the pharmaceutical composition, by weight, comprises 22 parts of umbilical cord MSC exosomes and 0.003 parts of yam saponin.

[0010] Furthermore, the pharmaceutical composition, by weight, comprises 17 parts of umbilical cord MSC exosomes and 0.005 parts of yam saponin.

[0011] Furthermore, in the pharmaceutical composition, the umbilical cord MSC exosomes are prepared by a method comprising the following steps:

[0012] S1. Mix the extract containing umbilical cord MSC exosomes with lectin and phosphate buffer solution, stir, wash and filter to obtain a liquid containing exosomes. S2. The liquid obtained in step S1 is passed through the first stage and the second stage of tangential flow ultrafiltration in sequence. The molecular weight cutoff of the first stage ultrafiltration membrane is 550kD and the molecular weight cutoff of the second stage ultrafiltration membrane is 600kD to obtain the exosome pre-purified solution. S3. Add the sucrose aqueous solution to the pre-purified exosome solution and perform density gradient centrifugation, then filter to obtain a filtrate containing exosomes; S4. Perform mixed-mode chromatography with a molecular weight cutoff of 400kD on the filtrate obtained in step S3 and collect the flow-through. S5. The flow-through solution obtained in step S4 is purified by passing it through a hydroxyapatite purification column and an affinity chromatography column to obtain the umbilical cord MSC exosomes.

[0013] On the other hand, a pharmaceutical formulation is also provided, comprising the pharmaceutical composition described in this invention and a pharmaceutically acceptable carrier.

[0014] Furthermore, the pharmaceutical preparation is an inhalation preparation.

[0015] Finally, the use of the pharmaceutical compositions or pharmaceutical preparations described in this invention in the preparation of medicaments for treating or improving chronic obstructive pulmonary disease is also provided.

[0016] Furthermore, the pharmaceutical composition or formulation described in the application treats or improves chronic obstructive pulmonary disease by reducing the expression levels of IL-1β, IL-6, and TNF-α in the lungs.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) For the first time, umbilical cord MSC exosomes were used in combination with yam saponin. Experiments showed that within a specific ratio range, the two had a significant synergistic effect in improving lung function indicators and reducing lung inflammation in animals with chronic obstructive pulmonary disease (COPD). The effect was clear and superior to that of a single component.

[0018] (2) For chronic obstructive pulmonary disease, a chronic respiratory disease with high incidence and high mortality, a novel drug composition solution based on cell-free therapy (exosomes) combined with natural active ingredients is provided, which has important clinical development value.

[0019] (3) A rapid purification method for umbilical cord MSC exosomes is provided. The method has clear operation steps, shortens the purification time to about 3 hours, doubles the efficiency of traditional methods, and has a recovery rate of up to 92%. Attached Figure Description

[0020] Figure 1 The figure shows the results of lung dynamic compliance (Cydn) measurement in COPD model mice under different drug effects.

[0021] Figure 2 The figure shows the results of airway resistance (RI) measurements in a COPD model mouse under different drug effects. Detailed Implementation

[0022] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0023] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0024] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0025] Yamosaponin, CAS No. 512-06-1, purity ≥98%.

[0026] In this invention, the umbilical cord MSC exosomes are exosomes derived from umbilical cord mesenchymal matrix cells.

[0027] Example 1 This embodiment describes a rapid purification method for obtaining umbilical cord MSC exosomes, including the following steps: S1. Add 20g of lectin to 100g of the extract containing umbilical cord MSC exosomes obtained by conventional methods, and stir at a rate of 290r / min for 3min; then add 40g of phosphate buffer solution (NaH2PO4, mass ratio 1:1). The Na2HPO4 mixture was stirred at a rate of 420 r / min for 7 min, and then washed and filtered twice to obtain a liquid containing exosomes. S2. The exosome-containing liquid from step S1 is passed into a first-stage tangential flow ultrafiltration device. The pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device is 0.22 μm. The injection pressure is controlled at 4 PSI, and the molecular weight cutoff of the ultrafiltration membrane is 550 kD. The ultrafiltration liquid is recovered to obtain permeate. Subsequently, the permeate is pumped into a second-stage tangential flow ultrafiltration device. The pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is 0.01 μm. The filtrate is recovered to obtain a pre-purified exosome solution. The injection pressure is controlled at 4 PSI, and the molecular weight cutoff of the ultrafiltration membrane is 600 kD. S3. Add 100g of 38% sucrose aqueous solution to the initial purified exosome solution, centrifuge at 100000g for 45min, and filter to obtain the filtrate containing exosomes.

[0028] S4. The exosome-containing filtrate obtained in step S3 is subjected to Capto Core 700 mixed membrane chromatography. The flow rate during the mixed mode chromatography process is 5 cm / min, the chromatography time is 120 min, the molecular weight cutoff is 400 kD, and the flow-through is collected.

[0029] S5. Add 50g of hydroxyapatite to the purification column, then add the flow-through solution obtained in step S4 to the purification column, stir and mix at a rate of 400 r / min, and incubate with shaking for 50 min at a speed of 150 rpm; transfer to an affinity chromatography column. The chromatography conditions are as follows: the equilibration buffer is 0.1 mol / L Tris. HCl, 0.3 mol / L NaCl, pH 7.5; eluent 0.02 mol / L TrisHCl, 0.4 mol / L NaCl, 0.6 mol / L imidazole, pH 7.9; equilibrate two columns, elute one column at a flow rate of 10 mL / min; wash once with 30% sodium dihydrogen phosphate solution, and allow to stand to obtain the exosome solution.

[0030] The exosome solution prepared by this purification method achieved a recovery rate of 92% and a purification time of 3 hours, which is twice as efficient as the traditional purification method in the prior art, which takes 6 hours. The purified exosomes were positive for CD63 / CD81 markers and had a particle size of D50 = 135 ± 15 nm.

[0031] Example 2 This embodiment describes the preparation of a composition containing umbilical cord MSC exosomes.

[0032] Weigh out 30 parts of exosomes prepared in Example 1, 0.001 parts of yam saponin, 0.2 parts of carbomer, 3 parts of mannitol, and 66.785 parts of physiological saline. Mix the above components evenly to obtain exosome composition No. 1.

[0033] Example 3 This embodiment describes the preparation of a composition containing umbilical cord MSC exosomes.

[0034] Weigh 27 parts of exosomes prepared in Example 1, 0.002 parts of yam saponin, 0.2 parts of carbomer, 3 parts of mannitol, and 69.798 parts of physiological saline. Mix the above components evenly to obtain exosome composition No. 2.

[0035] Example 4 This embodiment describes the preparation of a composition containing umbilical cord MSC exosomes.

[0036] Weigh 22 parts of exosomes prepared in Example 1, 0.003 parts of yam saponin, 0.2 parts of carbomer, 3 parts of mannitol, and 74.797 parts of physiological saline. Mix the above components evenly to obtain exosome composition No. 3.

[0037] Example 5 This embodiment describes the preparation of a composition containing umbilical cord MSC exosomes.

[0038] Weigh 17 parts of exosomes prepared in Example 1, 0.005 parts of yam saponin, 0.2 parts of carbomer, 3 parts of mannitol, and 79.795 parts of physiological saline. Mix the above components evenly to obtain exosome composition #4.

[0039] Example 6 This embodiment describes the preparation of a composition containing umbilical cord MSC exosomes.

[0040] Weigh 15 parts of exosomes prepared in Example 1, 0.007 parts of yam saponin, 0.2 parts of carbomer, 3 parts of mannitol, and 81.793 parts of physiological saline. Mix the above components evenly to obtain the No. 5 exosome composition.

[0041] Example 7 This embodiment describes the preparation of a composition containing umbilical cord MSC exosomes.

[0042] Weigh out 11 parts of exosomes prepared in Example 1, 0.009 parts of yam saponin, 0.2 parts of carbomer, 3 parts of mannitol, and 85.791 parts of physiological saline. Mix the above components evenly to obtain exosome composition No. 6.

[0043] Example 8 This embodiment describes the preparation of a composition containing umbilical cord MSC exosomes.

[0044] Weigh out 9 parts of exosomes prepared in Example 1, 0.011 parts of yam saponin, 0.2 parts of carbomer, 3 parts of mannitol, and 87.789 parts of physiological saline. Mix the above components evenly to obtain the No. 7 exosome composition.

[0045] Contrast agent 1 The difference between this embodiment and Embodiment 5 is that it does not contain yam saponin.

[0046] Weigh 17 parts of exosomes prepared in Example 1, 0.2 parts of carbomer, 3 parts of mannitol, and 79.8 parts of physiological saline. Mix the above components evenly to obtain the No. 1 exosome composition contrast agent.

[0047] Contrast agent 2 The difference between this embodiment and Embodiment 5 is that it does not contain exosomes.

[0048] Weigh out 0.005 parts of yam saponin, 0.2 parts of carbomer, 3 parts of mannitol, and 79.8 parts of physiological saline. Mix the above components evenly to prepare the No. 2 exosome composition contrast agent.

[0049] Example 9 This embodiment evaluates the therapeutic effects of different exosome compositions on COPD.

[0050] 9.1 Establishing an animal model of COPD Ninety-six male SPF-grade Wistar rats, weighing approximately 140-160g, were acclimatized for one week at 20-24℃ and 45%-65% relative humidity with alternating 12h / 12h light and dark conditions. They were randomly divided into four groups: control group, positive control group, control group 1, control group 2, model group, and test groups 1, 2, 3, 4, 5, 6, and 7, with eight rats in each group. Except for the control group, all other groups underwent modeling. On day 1 and day 14 of modeling, 20μL of 4mg / mL bacterial LPS was administered via intravenous drip. For the remaining time, the animals were placed in a fumigation chamber where 10 cigarettes were lit for 1.5 hours continuously for 30 days. On the second day of modeling, drug treatment was administered. The positive group received an intraperitoneal injection of dexamethasone solution (4 mg / kg). The control groups 1 and 2 received nebulized inhalation of exosome composition contrast agent 1 and exosome composition 2 via the Melton Inhalogic Nies Nose-only inhalation exposure system. The experimental groups 1-7 received exosome compositions 1-7 respectively. The model group received an equal volume of distilled water. The drug dosage was 0.25 mL / kg. Modeling and drug administration lasted for one month.

[0051] During the experiment, the animals' general condition was closely observed daily after model establishment and drug administration, including mental activity, respiration, and fur color. In the control group, rats were in good spirits, active, with glossy fur, and ate and drank normally. During the modeling period, animals in the model group were lethargic, had dull fur, and exhibited varying degrees of coughing and sneezing; severe cases showed rapid breathing, increased nasal discharge, and significant weight loss. In contrast, the positive control group and experimental groups 2-5 showed significant improvement compared to the model group; their fur became glossy, their spirits gradually recovered, and coughing and sneezing symptoms were alleviated. The control group 1, experimental group 1, and experimental groups 6 and 7 showed improvement compared to the model group, with significant relief of coughing and sneezing symptoms, but their mental state remained poor; the control group 2 showed no significant improvement compared to the model group.

[0052] After the last administration, the dynamic compliance (Cydn) and airway resistance (RI) of rats were measured using the DSI / BUXCO airway resistance and lung compliance monitoring system to evaluate the lung function of each group. The results are shown in Table 1. Figure 1 and Figure 2 As shown.

[0053] Table 1. Effects of different drugs on lung function in COPD model mice

[0054] Higher Cydn values ​​and lower RI values ​​indicate better lung function recovery. The model group had the worst indicators (Cydn=0.229, RI=0.501), while the control group had the best (Cydn=0.515, RI=0.318), serving as the baseline. Table 1 shows that the Cydn and RI of rats in experimental group #4 were 0.455 and 0.349, respectively, representing the best lung function recovery among all experimental groups. Furthermore, the Cydn value of experimental group #4 was slightly higher than that of the positive control group (0.452), while the RI was lower than that of the positive control group (0.355), closer to the level of the normal control group, indicating that the efficacy of the drug combination in experimental group #4 was slightly better than that in the positive control group. In experimental group #5, the rats had a Cydn value of 0.448 and an RI of 0.363, second only to experimental group #4, essentially on par with the positive control group, and significantly better than other experimental groups and the control group. The effect of control group #1 (without yam saponin) was significantly worse than that of experimental groups #4 and #5. There was no statistically significant difference between control group #2 and the model group, indicating that exosomes and yam saponin synergistically repaired lung function in rats. Within a mass ratio of 27:0.002-17:0.05, umbilical cord MSC-containing exosomes and yam saponin exhibited a synergistic effect. The combination of umbilical cord MSC-containing exosomes and yam saponin significantly improved lung function in COPD model rats, increased Cydn values, and decreased RI values, demonstrating a clear therapeutic effect.

[0055] After the last administration, lung tissue was collected from two rats in each group. Total RNA was extracted from the lung tissue using Trizol reagent on ice. After quantitative analysis, an equal amount of RNA was reverse transcribed into cDNA using a reverse transcription kit. An equal amount of cDNA was then used for PCR amplification. Finally, the cDNA was detected according to the instructions of the quantitative real-time PCR kit. The reaction conditions were: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 15 seconds, 60℃ annealing for 30 seconds, and 72℃ extension for 30 seconds, for a total of 38 cycles. The relative expression levels of IL-1β, IL-6, and TNF-α were calculated using the 2-ΔΔCt method, and the results are shown in Table 2.

[0056] Table 2 Results of inflammatory factor assay in lung tissue of rats in different experimental groups

[0057] Table 2 shows that in experimental group #4, the expression levels of the three inflammatory factors were the lowest: IL-1β (58.34), IL-6 (75.62), and TNF-α (305.34), close to the normal control group. Furthermore, the expression levels of inflammatory factors were lower than those in the positive control group, indicating that the anti-inflammatory effect of the drug composition in experimental group #4 was superior to that in the positive control. In control group #1, the anti-inflammatory effect of the drug composition without yamsaponin was significantly weaker than that in experimental groups #4 and #5. In control group #2, the inflammatory factor levels of the drug composition without exosomes were slightly lower than the model, indicating that this group of drug compositions had some anti-inflammatory effect, but the effect was limited. The levels of inflammatory factors in the drug composition of experimental groups 2-5 were significantly lower than those in other experimental groups and the control group 1-2, indicating that the combination of umbilical cord MSC exosomes and yam saponins in a mass ratio of 27:0.002-17:0.05 has a synergistic therapeutic effect on COPD model rats, significantly increasing Cydn, decreasing RI, improving lung function, significantly reducing the expression of IL-1β, IL-6, and TNF-α, and inhibiting lung inflammation. Among them, the drug composition of experimental group 4, with a mass ratio of umbilical cord MSC exosomes to yam saponins of 17:0.005, showed the best anti-inflammatory effect, followed by experimental group 5.

[0058] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A pharmaceutical composition for treating chronic obstructive pulmonary disease, characterized in that, This includes umbilical cord MSC exosomes and yam saponins.

2. The pharmaceutical composition according to claim 1, characterized in that, By weight, it includes 9-30 parts of umbilical cord MSC exosomes and 0.001-0.011 parts of yam saponin.

3. The pharmaceutical composition according to claim 2, characterized in that, By weight, it includes 27-17 parts of umbilical cord MSC exosomes and 0.002-0.005 parts of yam saponin.

4. The pharmaceutical composition according to claim 3, characterized in that, By weight, it includes 22 parts of umbilical cord MSC exosomes and 0.003 parts of yam saponin.

5. The pharmaceutical composition according to claim 3, characterized in that, By weight, it includes 17 parts of umbilical cord MSC exosomes and 0.005 parts of yam saponin.

6. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The umbilical cord MSC exosomes were prepared by a method comprising the following steps: S1. Mix the extract containing umbilical cord MSC exosomes with lectin and phosphate buffer solution, stir, wash and filter to obtain a liquid containing exosomes. S2. The liquid obtained in step S1 is passed through the first stage and the second stage of tangential flow ultrafiltration in sequence. The molecular weight cutoff of the first stage ultrafiltration membrane is 550kD and the molecular weight cutoff of the second stage ultrafiltration membrane is 600kD to obtain the exosome pre-purified solution. S3. Add the sucrose aqueous solution to the pre-purified exosome solution and perform density gradient centrifugation, then filter to obtain a filtrate containing exosomes; S4. Perform mixed-mode chromatography with a molecular weight cutoff of 400kD on the filtrate obtained in step S3 and collect the flow-through. S5. The flow-through solution obtained in step S4 is purified by passing it through a hydroxyapatite purification column and an affinity chromatography column to obtain the umbilical cord MSC exosomes.

7. A pharmaceutical preparation, characterized in that, It includes the pharmaceutical composition according to any one of claims 1-6, and a pharmaceutically acceptable carrier.

8. The pharmaceutical preparation according to claim 6, characterized in that, The drug formulation is an inhaled formulation.

9. The use of the pharmaceutical composition according to any one of claims 1-5 or the pharmaceutical preparation according to any one of claims 6-7 in the preparation of a medicament for treating or improving chronic obstructive pulmonary disease.

10. The application according to claim 9, characterized in that, The pharmaceutical composition or formulation treats or improves chronic obstructive pulmonary disease by reducing the expression levels of IL-1β, IL-6, and TNF-α in the lungs.

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